Hypoxia promotes oxidative base modifications in the pulmonary artery endothelial cell VEGF gene.
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Animals↗
Biomedical subjects
S P Ledoux
Publications and source records attributed to S P Ledoux.
Glial cell-specific differences in response to alkylation damage.
Oligodendrocytes are preferentially sensitive to the toxic, carcinogenic, and teratogenic effects of methylnitrosourea (MNU). The mechanisms responsible for this enhanced sensitivity have not been fully elucidated. One of the most vulnerable cellular targets for this chemical is mitochondrial DNA (mtDNA). To determine if differences in mtDNA damage and repair capacity exist among the different CNS glial cell types, the effects of MNU exposure on oligodendroglia, astroglia, and microglia cultured separately from neonatal rat brain were compared. Quantitative determinations of mtDNA initial break frequencies and repair efficiencies showed that whereas no cell type-specific differences in initial mtDNA damage were detected, mtDNA repair in oligodendrocytes, oligodendrocyte progenitors, and microglia was significantly reduced compared to that of astrocytes. In astrocytes, and all other cell types previously evaluated in our laboratory, >60% of N-methylpurines were removed from the mtDNA by 24 hr. In contrast, only 35% of lesions were removed from mtDNA of oligodendrocytes, oligodendrocyte progenitors, and microglia during the same time period. Mitochondrial perturbations by a variety of xenobiotics have been linked to apoptosis. In the present study, apoptosis, as determined by DNA laddering and ultrastructural analysis, was clearly induced by MNU treatment of cultured oligodendrocyte progenitors and microglia, but not in astroglia. These data demonstrate a correlation between diminished mtDNA repair capacity and the induction of apoptosis. However, further experimentation is necessary to determine if a causal relationship exists and contributes to the vulnerability of oligodendroglia following exposure to N-nitroso compounds in the environment or in chemotherapeutic regimen.
Alkylating Agents↗
Repair of O6-methylguanine in rat pancreatic beta-cells after exposure to N-methyl-N-nitrosourea.
Because of the possible involvement of O6-methyldeoxyguanosine as a cytotoxic and carcinogenic lesion in pancreatic beta-cells, studies were undertaken to assess the ability of rat beta-cells to repair this DNA lesion. Primary cultures of neonatal rat beta-cells were shown to contain very low levels of O6-methylguanine-DNA-methyltransferase activity, the predominant mechanism for repairing O6-methyldeoxyguanosine in mammalian cells. However, using a 32P-endlabeling assay to measure O6-methyldeoxyguanosine in cells after exposure to N-methyl-N-nitrosourea, it was determined that rat beta-cells repaired O6-methyldeoxyguanosine to a substantial extent over a 24-h period. To elucidate the mechanism of O6-methyldeoxyguanosine repair in the virtual absence of constitutive O6-methylguanine-DNA-methyltransferase expression, studies were performed to determine if O6-methylguanine-DNA-methyltransferase expression was enhanced in N-methyl-N-nitrosourea-treated beta-cells. No increase in O6-methylguanine-DNA-methyltransferase activity was detected 24 or 48 h after exposure. However, Northern blot analysis showed a two- to threefold elevation in O6-methylguanine-DNA-methyltransferase messenger RNA levels in beta-cells 12 and 24 h after N-methyl-N-nitrosourea treatment. This finding is the first demonstration of a change in O6-methylguanine-DNA-methyltransferase messenger RNA levels in a cell type with low constitutive activity.
Animals↗
Effects of streptozotocin on a clonal isolate of rat insulinoma cells.
Cytotoxic effects and DNA damage caused by streptozotocin, a potent beta-cell toxin and an important chemotherapeutic agent, in an insulin-secreting clonal isolate of a rat insulinoma cell line were evaluated. Cytotoxicity was monitored by phase-contrast microscopy and measurement of insulin release into the culture medium. DNA damage and repair were assessed by changes in nucleoid sedimentation rates. The insulinoma cells were resistant to streptozotocin toxicity as compared to normal rat beta-cells. They were also resistant to the stimulatory effects of glucose on insulin release. However, streptozotocin did cause DNA damage that was both dose- and time-dependent. Comparative analysis of streptozotocin-induced DNA damage and that produced by the aglycone N-methyl-N-nitrosourea revealed greater damage with the latter. Thus, streptozotocin, like N-methyl-N-nitrosourea, may enter these cells by passive diffusion rather than selective transport. DNA repair studies indicate that the nicks caused by streptozotocin are sealed and that the DNA is again supercoiled by 14 h. Therefore, overt toxicity may be avoided by a decreased drug uptake compared to normal beta-cells and efficient repair mechanisms. These studies suggest that an active glucose-sensing mechanism is necessary to enhance streptozotocin cytotoxicity in both normal and neoplastic beta-cells.
Adenoma, Islet Cell↗